AU735602B2 - Antifungal biocontrol agents, a process for preparing and treating the same - Google Patents

Description

A WO 98/35017 PCT/KR98/00015 1 ANTIFUNGAL BIOCONTROL AGENTS, A PROCESS FOR PREPARING AND TREATING THE SAME FIELD OF THE INVENTION The present invention relates to antifungal biocontrol agents, and to a process for preparing and treating the same. In particular, the present invention relates to two antifungal biocontrol strains of Streptomyces sp., WYE 20 and WYE 324, capable of enhancing plant growth and reducing plant diseases by suppressing fungal pathogens colonized in rhizosphere soils of host plants, and the preparation method and application method thereof.

BACKGROUND OF THE INVENTION It is well known that soilborne fungal phytopathogens cause enormous economic losses in the agricultural and horticultural industries. In particular, Rhizoctonia solani is one of the major fungal phytopathogens exhibiting strong pathogenicity; it is associated with seedling diseases as well as foliar diseases such as seed rot, root rot, damping-off, leaf and stem rot of many plant species and varieties, resulting in enormous economic losses. For example, Rhizoctonia solani AG 1 (IB) causes plant diseases in crops such as cucumber (Cucumis sativus and pepper (Capsicum annuum as well as brown patches of golf green creeping bentgrass, Rhizoctonia solani AG 2-2 causes large patches in fairway turfgrass on large areas of golf courses resulting in enormous economic losses. In addition, Phytophihora capsici is a widespread and highly destructive soilborne fungal phytopathogen that causes root and crown rot as well as the aerial blight of leaves, fruit, and the stems of peppers (Capsicum annuum It is very difficult to suppress Phytophthora blight in pepper plants if they are infected by a Phytophthora capsici. Phytophthora capsici is a particularly destructive phytopathogen responsible for blight of pepper plants during hot and humid rainy seasons resulting in the killing of the pepper plants and consequently enormous economic losses. As mentioned above, both Rhizoctonia solani and Phytophthora WO 98/35017 PCT/KR98/00015 2 capsici are major fungal phytopathogens which exhibit strong pathogenicity and produce spores which survive for a long time in harsh conditions. Thus, they repeatedly cause plant diseases in large areas when conditions for incidence of the disease are optimal.

Therefore, growers generally apply a mixture of fungicides to plants on a regular basis to control plant diseases caused by both Rhizoctonia solani and Phytophthora capsici. However, it is becoming increasingly difficult to control Phytophthora blight of pepper plants and Rhizoctonia brown patches of creeping bentgrass because of the emergence of strains of fungal phytopathogens resistant to fungicides. It is also becoming increasingly difficult to control these diseases due to the widespread incidence of Rhizoctonia solani resistant to fungicides in golf courses and the proliferation of Phytophthora capsici resulting from the successive cultivation of pepper plants.

Intensive use of agrochemicals has also provoked residual toxicity and environmental problems. Furthermore, agrochemicals are considered ineffective in controlling soilborne plant diseases due to wash out, and the lack of an efficient application which allows effective penetration into rhizosphere soils. It is also very difficult to expect long term protection of plants with an application of chemical fungicides. Therefore. using certain rhizosphere bacteria as a biocontrol agent can provide not only more effective and economical practices for the control of plant diseases such as Phytophthora blight caused by Phytophthora capsici and golf course turfgrass diseases caused by Rhizoctonia solani, but also increased environmental conservation.

It has been shown that use of antagonistic microorganisms is an attractive way to control fungal pathogens (Suh, Ph.D. Dissertation. University of Idaho, Idaho, USA, 1992; Crawford et al, Appl. Environ. Microbiol. 59:3899-3905, 1993; U.S. Pat. No. 5,403,584). The inventors disclosed in the prior art that certain Streptomyces species strains can be used to control plant pathogens by using a delivery medium containing peat moss, sand, and cornmeal, in the potting mixture/soil or by coating plant seeds with sodium alginate containing the CE01039005.4 3 biocontrol agent, resulting in the colonizing of the roots Pat. No. 5,403,584). It has also been disclosed in the prior art that peat containing a beneficial biocontrol agent, can be used to control plant pathogens Pat. No. 4,595,589). The delivery medium methods in the prior arts are suitable for the application of seedlings/potting mixtures, but are not quite suitable for direct application to plant seeds or plant roots to achieve efficient protection against fungal phytopathogens.

The object of the present invention is to provide novel strains of Streptomyces species which are capable of controlling fungal phytopathogens by direct application to plant seeds or plant roots utilizing specially designed formulations comprising delivery media containing a biologically pure culture selected from the newly isolated strains of Streptomyces species.

Further, it is an object of the present invention to provide antifungal biocontrol agents and describe their use in protecting plants from infections caused by soilborne fungal phytopathogens.

Summary of the invention The present invention has been achieved through the isolation of Streptomyces sp. WYE and WYE 324 which are capable of protecting plants against Rhizoctonia solani and Phytophthora capsici. The strains, Streptomyces sp. WYE 20 and WYE 324, are effective in S preventing the incidence of fungal diseases in plants and in enhancing plant growth in Sgreenhouse and agricultural field trials. Therefore, in one aspect of the present invention there is 20 provided microbially pure cultures of Streptomyces sp. WYE 20 and WYE 324 having the identifying characteristics of KCTC 0341BP and KCTC 0342BP respectively.

The present invention also provides for an antifungal biocontrol agent including a microorganism of a biologically pure culture of Streptomyces sp. WYE 20 and WYE 324.

In another aspect of the present invention there is provided a method of manufacturing 25 an antifungal biocontrol agent comprisinges an incubation process for the production of a biologically pure culture of Streptomyces sp. WYE20 or WYE324 and a downstream process comprising lyophilization of the harvested pure culture of Streptomyces sp. WYE 20 OR WYE CE01039005.4 4 According to another aspect of the present invention there is provided a method of manufacturing an antifungal biocontrol agent comprising an incubation process for the production of a biologically pure culture of Streptomyces sp. WYE20 or WYE324 and a downstream process comprising incorporation of the harvested pure culture into a proper delivery medium.

The present invention also provides a method of manufacturing an antifungal biocontrol agent wherein the method comprises the steps of: preparation of delivery medium consisting of to 65 w/w% of wheat bran, 1 to 5 w/w% of chitosan, 30 to 55 w/w% of wood sawdust, 1 to 3 w/w% of chitin, and 1 to 3 w/w% of Pharmamedia on the basis of the total weight of the delivery medium;autoclaving the resulting delivery medium; incorporation of Streptomyces sp.

WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) into the delivery medium; incubating incorporated cells of Streptomyces sp WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) at 25 0 C to 33 0 C for 5 to 14 days; and aseptically drying the resulting product.

According to a further aspect of the invention there is provided a method of manufacturing an antifungal biocontrol agent wherein the method comprises the steps of: preparation of delivery medium consisting of 1.0 to 3.0 w/w% of pectin, 0.1 to 0.6 w/w% of colloidal chitin, and the remaining water; and autoclaving the resulting delivery medium; and incorporation of Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) into the delivery medium.

T

The present invention also encompasses the various delivery media suitable for the treatment of plant seeds, seedling beds/pots, or potting mixtures/soil with Streptomyces s.p.

WYE 20 and WYE 324. The delivery medium is very useful for carrying Streptomyces sp.

WYE 20 and WYE 324 to inhibit the plant diseases caused by fungal phytopathogens.

:0o. 25 In a particular embodiment according to the present invention, the delivery medium consists of wood sawdust, wheat bran, chitin, chitosan, and Pharmamedia (trade name; made by SThe Budkeye Oilseed Products Company, Texas, USA) together with Streptomyces sp. WYE ror WYE 324. In another particular embodiment, the delivery medium consists of pectin and 7 lloidal chitin and water together with Streptomyces sp. WYE 20 or WYE 324. In the preferred CE01039005.4 4a embodiment, the delivery medium comprises at least 105 colony forming units of Streptomyces sp. WYE 20 or WYE 324 per gram of delivery medium.

The present invention also encompasses methods for reducing fungal infection in seeds, seedlings and/or growing plants prior to or during sowing and growing seasons through the treatment of plant seeds, seedling beds/pots, potting mixture/soils, spraying or in-furrow application.

Detailed description of the embodiments According to the present invention, Streptomyces sp. WYE 20 and WYE 324 are provided that are effective in inhibiting fungal diseases and in enhancing the growth of plants such as pepper, cucumber, and turfgrasses, although their scope is not limited to these examples.

The present invention encompasses the various delivery media that are useful for carrying Streptomyces sp. WYE 20 and WYE 324 to inhibit the plant diseases caused by fungal phytopathogens.

In a particular embodiment of the present invention, the delivery medium consist of to 65 w/w% of wheat bran, 1 to 5 w/w% of chitosan, 30 to 55 w/w% of wood sawdust, 1 to 3 w/w% chitin, and 1 to 3 w/w% of Pharmamedia on the basis of the total weight of the delivery medium. In the preferred embodiment, the delivery medium further consists of 0.2 to 3.5 w/w% of sporulation medium as an additional component on the basis of the total weight of the delivery medium. The preferred sporulation medium is selected from ATCC #5 sporulation medium or yeast extract-glucose-mineral salts, but the presentation does not exclude other S. sporulation media.

In another particular embodiment, the delivery medium consists of 1.0 to 3.0 w/w% of pectin and 0.1 to 0.6 w/w% of colloidal chitin in water.

-WO 98/35017 PCT/KR98/00015 In the preferred embodiment, the delivery medium comprises 10' to 100, preferably 10 7 to 10 8 colony forming units of Streptomyces sp. WYE 20 or WYE 324 per gram of delivery medium.

The present invention includes a process for the production of mycelium and spore inocula of Streptomyces sp. WYE 20 and WYE 324. Viable cells of Streptomyces sp. WYE 20 and WYE 324 are produced by incubating vegetative cells in the yeast extract-glucose-mineral salts or modified bennett liquid medium under the proper conditions.

A process for preparing an antifungal biocontrol agent comprises as follows; Streptomyces sp. WYE 20 or WYE 324 are prepared by incubating at 130 rpm to 300 rpm, at a temperature of 25 °C to 33 °C for 3 to 7 days.

After harvesting cells of Streptomyces sp. WYE 20 or WYE 324, the cells are lyophilized or mixed directly into a delivery medium.

Further, according to the present invention, a process for preparing an antifungal biocontrol agent comprises the following steps; preparing a delivery medium containing 40 to 65 w/w% of wheat bran. 1 to 5 w/w% of chitosan, 30 to w/w% of wood sawdust, 1 to 3 w/w% of chitin and 1 to 3 w/w% of Pharmamedia on the basis of the total weight of the delivery medium; autoclaving the resulting delivery medium; incorporating Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP).into the delivery medium; incubating incorporated cells of Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) at 25 "C to 33 "C for 14 days; and aseptically drying the resulting product to obtain an antifungal biocontrol agent.

Preferably, the process further comprises aseptically blending the resulting dried product.

In the above process, the delivery medium is optionally pelletized and then coated with 0.2 to 3.5 w/w% of sporulation medium on the basis of the total -WO 98/35017 PCTIKR98/00015 6 weight of the delivery medium.

Streptomyces sp. WYE 20 or WYE 324 is incorporated to a concentration of 105-10, preferably 107-108 colony forming unit per gram of delivery medium.

Autoclaving of the delivery medium is carried out at 121 C for 30 to minutes.

In another embodiment of the present invention, a process is provided for preparing an antifungal biocontrol agent which comprises preparing a delivery medium consisting of 1.0 to 3.0 w/w% of pectin and 0.1 to 0.6 w/w% of colloidal chitin in water; autoclaving the resulting delivery medium; and incorporating Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) into the delivery medium.

In the above embodiment of the present invention, Streptonmyces WYE or WYE 324 is added to the delivery medium to a final concentration of 105- 10', preferably 107-108 colony forming unit per gram of delivery medium.

Further, the present invention relates to a method for treating the antifungal biocontrol agent which comprises coating, mixing, spraying or infurrow applicating the agent to plant seeds, potting mixture, plants, or soils thereof.

The present invention also relates to an antifungal biocontrol agent which comprises one of the newly isolated strains of Streptomyces sp. WYE 20 and WYE 324. Therefore, it is appreciated that an antifungal biocontrol agent containing microorganisms having an antifungal property equal to that of the strains together with the delivery medium according to the present invention is within the true spirit and scope of the present invention.

The present invention will be further described in the following examples.

However, the present invention is not limited to the following examples.

Materials and Methods 1. Culture Medium of Microorganism All bacterial culture media used distilled water and were sterilized by autoclaving prior to use. All bacterial samples were treated by standard aseptic -WO 98/35017 PCT/KR98/00015 7 laboratory techniques to maintain purity.

1) CYD (casamino acid/yeast extract/dextrose/agar) medium contains casamino acids (Difco: 0.5g/l), yeast extract (Difco: 0.8g/l), D-glucose (0.4 g/1),

K.HPO

4 (2.0g/1; pH and agar (18g/l) in distilled water.

2) WYE (water/yeast extract/agar) medium, modified from Reddi and Rao (1971) contains yeast extract (0.25g/1), K 2

HPO

4 (2.0g/l; pH 7.2-7.4) and agar (18g/l) in distilled water.

3) YGM (yeast extract/glucose/mineral salts) medium comprises 0.6% yeast extract (Difco Laboratories, Detroit, MI, glucose, and phosphate mineral salt solution (5.3g of Na 2

HPO

4 1.98g of KH 2

PO

4 0.2g of MgSO 4 7H 2 0, 0.2g of NaCI, 0.05g of CaCI 2 2H2O, plus 1.0 ml of trace elements (Pridham and Gottlieb, J. Bacteriology 56:107-114, 1948) per liter of deionized pH 7.1 to The solution of trace elements consists of 0.64g of CuSO.

2 0, 0.1 g of FeSO 4 7H 2 0, 0.79g of MnCl2 4H 2 0, 0.15g of ZnSO 4 7H 2 0 in 100 ml of distilled water.

4) CM (chitin/mineral salts/agar) medium comprises 0.4 to 0.6% (w/v) colloidal chitin prepared by the previously known method (Hsu and Lockwood, Applied Microbiology, p422-426, 1975), 0.6% (NH 4 2 SO4, and 2.0% (w/v) agar per liter of phosphate mineral salt solution described above; pH 7.0 to 7.2.

5) Laminarin agar medium comprises 0.25% laminarin, 0.6% (w/v)

(NH

4 2

SO

4 and 2.0% agar per liter of phosphate mineral salt solution described above; pH 7.2 to 7.4.

6) Modified Bennett liquid medium contains yeast extract beef extract peptone glucose (10g/1), and nystatin (5 pg/ml; pH 6.5-7.5) in distilled water.

7) Modified Bennett Agar medium contained yeast extract beef extract peptone glucose (10g/1), nystatin (5 /g/ml; pH and agar (20g/l) in distilled water.

8) ISP medium #2 (Difco).

9) ISP medium #3 (Difco).

WO 98/35017 PCT/KR98/00015 8 ISP medium #4 (Difco).

11) Sporulation medium (ATCC medium contains yeast extract beef extract tryptose FeSO 4 (0.01g/l), glucose and agar (15g/l) (17th Edition ATCC Catalogue of Bacteria and Bacteriophages).

2. Identification of Streptomyces sp. WYE 20 and WYE 324 Strains WYE 20 and WYE 324 were identified as Streptomyces species on the basis of the morphological, physiological, and chemical characteristics of the genus Streptomyces, as defined in Bergey's Manual of Systematic Bacteriology (1989); the International Streptomyces Project (ISP) (1974)): and Williams, et al. (1983). The summarized results of these strains in the respects of morphological, physiological, and chemical characteristics are reported in Tables I to V.

3. Preparation of seed culture and storage of Streplomyces sp. WYE and WYE 324 Spores from a CYD agar slant were inoculated with an inoculation loop into 500ml flasks containing 50ml of modified bennett liquid medium (pH 6.5-7.5).

The medium was sterilized by autoclaving for 15 minutes at 121 "C prior to inoculation. The inoculated flasks were then incubated with shaking at 130 to 300 rpm at 25 °C to 33 °C for 1-4 days to provide a standard inoculum.

For short-term use. Streptomyces sp. WYE 20 and WYE 324 were incubated on CYD or WYE agar slant at 25 °C until sporulated and then stored at 4 °C until used. The cultures were transferred every 4 weeks. Spores suspended in autoclaved glycerol (121 15 minutes) were used for long-term storage at -70 °C.

4. Harvesting and production of mycelia and spores in liquid culture One liter flasks containing 200ml of modified bennett liquid medium or 200 ml of YGM (pH 6.5-7.5) medium were inoculated with 8ml of each seed culture for the production of viable cells of mycelia and spores of Streptomyces sp. WYE 20 and WYE 324. The inoculated flasks were incubated with shaking at 130 to 300 rpm at 25 °C to 33 °C for 3 to 7 days. Viable cells of mycelia and -WO 98/35017 PCT/KR98/00015 9 spores produced through the above process were aseptically harvested by centrifugation at 4,000 rpm for 10 minutes.

Production of viable cells of mycelia and spores with delivery medium and preparation of antifungal biocontrol agent.

Preparation of antifungal biocontrol agent in the form of powder comprising Streptomyces sp. WYE 20 or WYE 324 and a delivery medium The delivery medium of the present invention which consisted of wheat bran of 40 to 65 chitosan of 1 to 5 wood sawdust of 30 to 55 w/w%, chitin of 1 to 3 Pharmamedia (The Budkeye Oilseed Products Company, Fortworth, Texas, of 1 to 3 w/w% on the basis of the total weight of the medium was thoroughly mixed. The resulting delivery medium was pelletized and spray-coated with sporulation medium (ATCC #5 or YGM medium) of 0.2 to and then autoclaved at 121 °C for 30 to 40 minutes. There was no effect on cellular growth when the sporulation medium was added below 0.2 whereas the initial cellular growth was retarded when the sporulation medium was added over 3.5 w/w%.

One hundred to two hundred ml of Streptomyces sp. WYE 20 and WYE 324 (10'-107 cfu/ml) were inoculated into the resulting autoclaved delivery medium and then incubated at 30 °C for 5 to 14 days. Streptomyces sp. WYE and WYE 324 in the delivery medium were aseptically harvested and dried in sterilized laminar flow bench. The dried product was aseptically blended to obtain an antifungal biocontrol agent comprising Streptomyces sp. WYE 20 or WYE 324 and the delivery medium.

Preparation of antifungal biocontrol agent comprising Streptomyces sp.

WYE 20 or WYE 324 and a liquid delivery medium.

Preparation of the antifungal biocontrol agent was carried out by incorporating viable cells of mycelia and spores of Streptomyces sp. WYE 20 or WYE 324 obtained in the above into autoclaved delivery medium of 1.0 to w/w% of pectin and 0.1 to 0.6 w/w% of colloidal chitin in water.

6. Fungal Pathogens _WO 98/35017 PCT/KR98/00015 The following fungal pathogens were used for an antifungal test: Pythium ultimum, Pythium graminicola, Rhizoctonia solani, Rhizoctonia solani AG 1 (IB), Rhizoctonia solani AG 2-2 Fusarium oxysporum, Fusarium sambucinctum, Fusarium solani, Phytophthora capsici, Phytophthora parasitica, Sclerotinia sclerotiorum. Sclerotium cepivorum, and Verticillium dahliae. All strains were grown on potato dextrose agar (Difco) or corn meal agar (Difco) at 25 "C and stored at 4 "C.

7. Determination of Cell Number in Delivery Medium One gram of delivery medium containing Streptomyces sp. WYE 20 or WYE 324 was added to 9ml of sterile distilled water and thoroughly mixed with vortexTM. The resulting suspension was serially diluted and spread on CMA plates to determine colony forming unit (cfu) per gram. The plates were incubated at 30 °C and colony formation was observed. The same method was used to determine cfu/ml of delivery medium in the form of liquid.

8. In Vivo Bioassay to Determine Antifungal Activity of Sireplomvces sp.

WYE 20 and WYE 324 The activity of Streptomyces sp. WYE 20 or WYE 324 to enhance plant growth and to reduce plant diseases caused by fungal pathogens was determined by treating cucumber, pepper, or golf course turfgrasses with Streptomyces sp.

WYE 20 or WYE 324 in a delivery medium, and then planting treated and untreated seeds in a suitable growing medium or agricultural fields. The activity of Streptomyces sp. WYE 20 or WYE 324 in a delivery medium as a biocontrol agent was measured in terms of emergence, outgrowth of emerged plants, plant height, and capability of disease control.

EXAMPLE I Isolation of Streptomyces sp. WYE 20 and WYE 324 Soil samples were obtained from four different rhizosphere-associated soils in Goesan, Chungbuk Province, Korea. Isolation of strains was carried out by serial-dilution/spread-plate technique on WYE agar plates. The obtained -WO 98/35017 PCT/IM8/00015 11 strains were tested with respect to antagonistic activity against Rhizoctonia solani and Phytophthora capsici: enzyme activities exhibiting fungal cell wall degradation; root colonization, growth at a low temperature (4 8 and the possibility of large cell mass production to isolate two novel strains. The two novel strains were named Streptomyces sp. WYE 20 and WYE 324, respectively.

Purification of Streptomyces sp. WYE 20 and WYE 324 was carried out by incubating the strains on WYE plates at 25 °C for 4 to 10 days to allow the strains to sporulate, and then their colonies were isolated and streaked onto new WYE agar plates to obtain pure cultures. Pure cultures of Streptomyces sp. WYE and WYE 324 were transferred to CYD agar slants, incubated at 25 °C 30 °C until sporulated, and stored at 4 The cultures were transferred every 4 weeks.

EXAMPLE 11 Identification and Characterization of Streptomyces sp. WYE 20 and WYE 324 The morphological, physiological, and chemical characteristics of microorganisms obtained in Example I were investigated on the basis of Bergey's Manual of Systematic Bacteriology (1989). The results are shown in Tables I to

V.

Table I Cellular Aerial Substrate Color of Spore Medium Growth Mycelium Mycelium Mass WYE WYE WYE WYE WYE WYE WYE WYE 324 20 324 20 324 20 324 ISP #2 Good Good Abun Abun Light Light Grayish Grayish -dant -dant Yellow Yellow Pink Pink ISP #3 Good Good Abun Abun Pale Pale Grayish Grayish -dant -dant Yellow Yellow Pink Pink ISP #4 Good Good Abun Abun Pale Pale Grayish Grayish -dant -dant Yellow Yellow Pink Pink -WO 98/35017 WO98/3017PCT/KR98/00015 Table Ila Characteristics Strain WYE 20 Strain WYE 324 Spore chain morphology Spirales Spirales Spore chain ornamentation Smooth Smooth Spore shape Cylinderical Cylinderical Diffusible Melanin production (brown-dark brown) (brown-dark brown) Antimicrobial activity: Aspergillus niger Bacillus subtilis NCIB 361 0 Sireptomyces murinus Candida aihi cans Saceharomyce cerevisiae CBS 1172 Micrococcus lute us NCIB 196 Lecithinase activity Pectin hydrolysis Skim milk hydrolysis Starch hydrolysis Nitrate reduction

H-

2 5 production ±4wek NO 98/35017 ~WO 9835017PCT/K118/00015 Table Ila(continue)_________ Characteristics Strain WYE 20 Strain WYE 324 Antibiotic resistance (gug/ml): Oleandomycin (100) -1i11n1 giz -11111 TI Neomycin (50) Rifampicin (50) -2iiu giz Lincomycin (100) Novobiocin (100) Ganamycin. (100) -2ini giz -3111 giz Ampicilin (100) Streptomycin (100) -31111 Viz -2nii giz Kasugomycin (100) I 11111iz Tetracycl ine (100) Chloramphenicol (1 00) 3ii11 -growth no growth inhibition zone inhibition zone (giz) >_4_mm -WO 98/35017 -WO 9835017PCTIMS9/00015 Table 11b Characteristics Strain WYE 20 Strain WYE 324 Degradation activity: Xanthine Elastin Arbutin Xylan L-tyrosine Al lantoin Growth at 45 'C Growth at pH 4.3 Growth in the presence of chemical inhibitors w/v): NaCI Sodium azide (0.01) Phenol Potassium tellurite (0.001) Crystal violet (0.0001) Neolin Neolin Tween 20 Tween 20 Rizoraxrm 1) RizoraX TM Gochutan TM Smirax~ m 1) Lidomil MG TM (0.125) Lidomil MG TM (0.25) Angco

TM

(0.1) -~WO 98/35017 PTK9IO1 PCT/ER98/00015 Table Ilb(continue) Characteristics Strain WYE 20 Strain WYE 324 Nitrogen utilization 1% w/v): L-asparagine DL- a -amino-n-butyric acid L-cysteine L-valine L-phenylalanine L-histidine L-hydroxyproline L-arginine L-methionine Potassium nitrate L-serine L-threonine T" Trade mark -WO 98/35017 PCT/KR98OO0i5 16 Table Llc Characteristics Strain WYE 20 Strain WYE 3 24 Carbon utilization(l w/v): Sucrose Meso-inositol Mannitol L-Rhamnose Raffinose D-Melezitose Adonitol D-MeI ibiose Dextran A Xylitol L-Arabinose D-Fructose D-Galactose D-Glucose D-Salicin D-Xylose Sorbose D-Lactose D-Mannose Trehalose Maltose Cellobiose Inulin Sodium acetate 1) Sodium citrate 1) Sodium malonate 1) Sodium propionate, 1) Sodium pyruvate 1) Diaminopimelic acid LL

LL

Table III Streptomyces sp. WYE Fatty acid Content 1 2 3 4 Minimum Maximum Average Saturated fatty acid 14:0 0.33 0.30 0.53 0.53 0.30 0.53 0.42 15:0 1.52 1.54 1.13 1.19 1.13 1.54 1.35 16:0 5.09 5.22 5.95 6.03 5.09 6.03 5.57 17:0 0.30 0.31 nd nd nd Unsaturated fatty acid 16:1 cis 9 6.42 6.25 5.45 5.80 5.45 6.42 5.98 17:1 cis 9 1.08 1.06 0.65 0.63 0.63 1.08 0.86 Methyl group branch 13:0 anteiso 0.14 0.14 nd nd nd 14:0 iso 1.30 1.23 2.40 2.26 1.23 2.40 1.80 15:0 iso 7.19 7.09 7.58 7.21 7.09 7.58 7.27 15:0 anteiso 27.95 27.48 30.94 32.13 27.48 32.13 29.63 a> FattyacidStreptomyces sp. WYE Content 1 2 34 Minimum Maximum Average 16:1 iso H 2.80 2.69 3.01 2.98 2.69 3.01 2.87 16:0Oiso 15.01 15.17 17.71 17.09 15.01 17.71 16.25 160 H 3.4 3.42 2.77 2.69 2.69 3.42 3.08 17:1 anteiso C 7.24 7.25 5.56 5.82 5.56 7.25 6.47 17:0 iso 1.83 1.93 1.85 1.65 1.65 1.93 1.82 17:0 anteiso 11.78 12. 35 10.77. 10. 63 10.6-3 12.35 11.38 17:0 10CHI 0.23 0.27 nd nd nd 18:1 iso H 0.99 1.02 0.62 0.59 0.59 1.02 0.81 Hydroxyl group branch -17:0Oiso 20H 0.40 0.41 nd nd nd 17:0 30H 0.27 0.32 nd nd nd Cyclopropane__ 17:0 Cyclopropane 3.30 3. 27 2.30 2.23 2.23 3.30 2.78

CD

C)

C,

L,

Table III(continue) 0 Faty ad Streptonmyces sp. WYE Fatty acid Content 1 2 3 4 Minimum Maximum Average Unknown 17.595 SM 0.78 0.82 0.50 0.55 0.50 0.82 0.66 Note: The first and second time: Cells grown in Trypticase Soy Broth (TSB).

The third and fourth time: Cells grown on Trypticase Soy Agar (TSA).

no detection. nd: not determined.

Table IV Streptoinyces sp. WYE 324 Fatty acid Content 1 2 34 Minimum Maximum Average Saturated fatty acid 14:0 0.28 0.26 0.42 0.45 0.26 0.45 0.35 15:0 2.22 2.19 1.23 1.25 1. 2 3 2.22 1.72 16:0 4.'3 4 4.40 5.89 6.19 4.34 6.19 5.21 17:0 0.40 0.43 nd nd nd Unsaturated fatty acid 15:1 B 0.20 0.15 nd nd nd 16:1 cis 9 6.40 6.39 5.66 5.54 5.54 6.40 6.00 17:1 cis 9 1.40 11. 43 0.78 0.71 0.71 1.43 1.08 Methyl group branch 13:0Oiso 0.08 0.10 ndnd nd 13:0Oanteiso 10.13 10.14 1 I I nd nd nd IV(continue) Fatty acidSireptornyces sp. WYE 324 Content 1 2 3 4 Minimum Maximum Average 14:0 iso 1.64 1.62 2.18 2. 30 1.62 2.30 1.94 15:0 iso 7.48 7.48 8.15 8.10 7.48 8.15 7.80 15:0 anteiso .30. 25 3)0. 10 31.65 31. 76 30.10 31.76 30.9 4 16:1 iso H 2.59 2.57 3 34 3.23 2.57 -3.3 4 2.93 16:0Oiso 14.05 14.06 16.67 16.69 14.05 16.69 15.37 16:0 9? CH 3 3.56 3.56 3.29 3. 12 3).12 3.56 3.3 17:1 anteiso C 6.68 6.65 5.97 15.90 5.90 6.68 6.30 17:0Oiso 1.77 1.82 2.14 2.16 1.77 2.16 1.97 17:0Oanteiso 10.78 10.95 10.68 10.72 10.68 10.95 10.78 17:0O10CH 3 0.18 0.18 nd nd nd 18:1liso H 0.49 0.50 nd nd rid Hydroxyl group branch 17:0Oiso 20H 0.50 0.51 id nd nd

I

Table IV(continue) FattyacidStreptomyces sp. WYE 324 Content 1 2 3 4 Minimum Maximum Average 17:0 30H 0.24 0.19 nd nd nd Cyclopropane 17:0OCyclopropane 3.15 3.14 1.95 1.89 1.89 1.15 2.53 Unknown 17.595 SM 10.68 0.69 1 I nd nd nd Note: The first and second time: Cells grown in Trypticase Soy Broth (TSB).

The third and fourth time:'Cells grown on Trypticase Soy Agar (TSA).

-no detection. nd: not determined.

Table V Characteristics Sireptoniyces Sirepomnyces Streptoinyces Streptomyces sp. WYE 20 coloinbiensis sp. WYE 324 goshikiensis ATCC 27425 23914 Spore chain: Rectiflexibles Spore chain: Retinaculiaperti-- Spore chain: Spirales Spore chain: Verticillati_______ Spore surface ornamentation: Smooth Spore surface ornamentation: Rugose-- Color of spore mass: Red Color of spore mass: Gray Color of spore mass: Green Diffusible pigment: Red/Orange Diffusible pigment: Yellow/Brown Melanin production Fragmentation Use of DL- a -amino-n-butyric acid(0. w/v) Table V(continue) Characteristics Streptornyces Streptonzyces Streptornyces Streptomyces sp. WYE 20 coloinbiensis sp. WYE 324 goshikiensis 27425 ATCC 23 )914 Use of L-histidine w/v) Use of L-hydroxyprol ine w/v) Lecithinase activity Pectin hydrolysis Nitrate reduction H12S Production Bacillus subtilis NCIB Streptoinyces murinus Aspergillus niger Xanthin degradation activity Allantoin degradation activity Arbutin degradation activity Neornycin (50 p g/mI) resistance u g/mI) resistance--

I

Table V(continue) Characteristics Streptornyces Streptornyces Streptomyces Streptomyces sp. WYE 20 coloibiensis sp. WYE' 324 goshikiensis ATCC 27425 23914 Growth at 45 *C Growth at NaCI Growth at sodium azide (0.0 Growth at phenol w/v) Utilization of D-xylose w/v) Utilization of meso-inositol Utilization of mannitol Utilization of D-fructose w/v) Utilization of L-rhamnose w/v) Utilization of raffinose Utilization of innulin Utilization of adonitol Utilization of cellobiose w/v) WO 98/35017 PCT/KRM/00015 26 The two strains obtained in Example I were identified as the genus Streptomyces, as a result of analyzing the data of Tables I to V on the basis of Bergey's Manual of Systematic Bacteriology (1989); International Streptomyces Project (ISP) (1974)); and William et al (1983).

The two strains were named as WYE 20 and WYE 324. respectively. The strain WYE 20 might belong to the species Streptomyces colombiensis or a closely related species in cluster 61 on the basis of the morphological, physiological, and chemical characteristics of the genus Streptomyces, but the strain was identified as a noble strain of the genus Streptomyces. Strain WYE 324 might belong to the species Streptomyces goshikiensis or a closely related species in cluster 61 on the basis of the morphological, physiological, and chemical characteristics of the genus Streptomyces, but the strain was identified as a noble strain of the genus Streptomyces.

The strains WYE 20 and WYE 324 were deposited in the Korean Culture Type Collection (KCTC). Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea on June 18, 1997 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, assigned with the Accession No. KCTC 0341BP and KCTC 0342BP, respectively.

EXAMPLE III Optimum Temperature for The Cellular Growth of Streptomyces sp.

WYE 20 and WYE 324 To investigate the effect of temperature on the growth of the Streptomyces sp. WYE 20 and WYE 324, spores of strains WYE 20 and WYE 324 were streak-inoculated on modified bennett agar plates. The streaked plates were incubated at predetermined temperatures of 4 8 C, 27 C, 37 and 45 °C for 3 weeks. The growth was recorded at 7, 14, and 21 days. The results are shown as good growth slow growth no growth in Table VI.

WO 98/35017 PCT/KR98/00015 27 Table VI Strain 4 °C 8 °C 27 °C 37 °C 45 °C 7 21 7 14 7 14 7 14 7 14 days days days days days days days days days days WYE WYE 324 As shown in Table VI, strain WYE 20 represented slow growth after a week of incubation at 4 °C and good growth after 3 weeks' incubation, whereas strain WYE 324 represented slow growth after 3 weeks incubation at 4 Strain WYE 20 represented good growth at both 8 °C and 27 However, strain WYE 324 represented slow growth after a week of incubation at 8 °C and represented good growth after 2 weeks' incubation at 8 In addition, strain WYE 324 represented good growth at 27 Strains WYE 20 and WYE 324 represented slow growth after a week of incubation at 37 °C and represented good growth after 2 weeks incubation at 37 but they did not grow at 45 °C.

EXAMPLE IV Enzyme Activity and Antagonism Assay Enzyme Activity Assay To assay the enzyme activity of the strains WYE 20 and WYE 324. both chitinase and -1,3-glucanase were used which were known as enzymes capable of parasitizing/inhibiting fungal pathogens.

In order to determine the chitinase activity of strains WYE 20 and WYE 324, each strain was streaked on CM agar plates which contained colloidal chitin as a sole carbon source, and then incubated at 30 °C for 14 days. Colony formation and growth were observed. The strains WYE 20 and WYE 324 were proved to have a chitinase activity from the above.

WO 98/35017 PCT/KR98/00015 28 To determine 8 -1,3-glucanase activity of the strains WYE 20 and WYE 324, each strain was streaked on laminarin agar plates which contained laminarin as a sole carbon source, and then incubated at 27 °C for 7 days. Colony formation and growth were observed. The strains WYE 20 and WYE 324 were proved to have a 8 -1,3-glucanase activity from the above.

Antagonism Assay An in vitro plate assay was performed to test the antagonistic activity of strains WYE 20 and WYE 324 in terms of growth inhibition of fungal phytopathogens. Each strain was streak-inoculated on CMA plates and incubated at 30 °C for 6 to 10 days. A PDA (Potato Dextrose Agar) block containing actively growing fungal mycelia was then aseptically placed in the center of the plate and incubated at 25 °C for 24 to 192 hours. The above PDA block was inoculated into CMA plates not containing strain WYE 20 or WYE 324 and used as a control.

The bioassay was replicated on three plates. The antagonism was recorded as a degree of inhibition of mycelial growth of fungal pathogens as shown in Table VII.

.WO 98/35017 *WO 9835017PCT/KR98/00015 29 Table VII.

Antagonistic activity Fungal Pathogens WYE 20 WYE 324 Pythium ultimum' Pythium graminicola 2 Fusarium oxysporum Fusarium sambucincturn nt Fusarium solani Rhizoctonia solani Rhizoctonia solani AG 1 (113) Rhizocionia solani AG 2-2 (IV) Phybophthora capsici Phylophthora parasitica Scierotinia scierotiorum Scieroliurn cepivorum' Vert icilliuni dahliae' nt 24 hour incubation, 236 hour incubation, '192 hour incubation.

Remnant: 96 hour incubation Strong inhibition with zone of inhibition 1.0 cm Growth definitely retarded, with obvious zone of inhibition near colony Not antagonistic, or very weakly antagonistic.

not tested.

.0 As shown in Table VII, strains WYE 20 and WYE 324 showed an obvious zone of inhibition against Pythiurn ultimun. Strain WYE 20 showed strong antagonism against Pythium graminicola, Fusarium oxysporumn Fusarium -WO 98/35017 PCT/KR98/00015 sambucinctum, Fusarium solani, Rhizoctonia solani, Rhizoctonia solani AG 1 Rhizoctonia solani AG 2-2 Phytophthora capsici, Phytophthora parasitica, Sclerotinia sclerotiorum, Sclerotium cepivorum and Verticillium dahliae. In addition, strain WYE 324 also showed strong antagonism against Pythium graminicola, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Rhizoctonia solani AG 1 Rhizoctonia solani AG 2-2 Phytophthora capsici, Phytophthora parasitica, Sclerotinia sclerotiorum, and Sclerotium cepivorum.

EXAMPLE V Preparation of Antifungal Biocontrol Agent in the Form of Powder A delivery medium was prepared by mixing wheat bran, chitosan, wood sawdust, chitin, and Pharmamedia according to Table VIII. The delivery medium was pelletized using an extruder. In the process of pelletizing, the degree of pellet formation was determined and recorded as good in the case of good pellet formation without debris and disruption; whereas recorded as poor in the case of poor pellet formation. The results are shown in Table VIII. It was also determined as poor, moderate, and good according to the air penetration and cellular growth of the delivery medium. The results are shown in Table VIII.

One hundred fifty ml of the culture of Streptomyces sp. WYE 20 or WYE 324 (10'-107 cfu/ml) obtained in "Materials and Methods" were inoculated into the resulting autoclaved delivery medium and then incubated at 30 "C for 5 to 14 days.

The cultures of Streptomyces sp. WYE 20 or WYE 324 in each delivery medium were aseptically harvested and dried in a sterilized laminar flow bench. The dried product was aseptically blended to obtain an antifungal biocontrol agent consisting of Streptomyces sp. WYE 20 or WYE 324 and the delivery medium, in the form of powder.

The number of cells of WYE 20 or WYE 324 in the delivery medium was adjusted to 105-10" cfu/g delivery medium. The resulting antifungal biocontrol agent which consisted of WYE 20 or WYE 324 and a delivery medium was kept WO 98/35017 PCT/KR98/00015 31 at 4 "C and 25 °C for 3 months. The viability and growth of WYE 20 or WYE 324 in the delivery medium was determined at intervals of one month and recorded at 3 months.

One gram of delivery medium containing WYE 20 or WYE 324 was added to 9ml of sterile distilled water and thoroughly mixed by vortax. The cfu per gram of the resulting suspension was determined by serial-dilution and spreadplate technique and shown in Tables VIII and IX.

Table VIII (unit: w/w%) Delivery Medium Wheat bran Chitosan Wood Chitin Pharma Pellet Air Cellular sawdust media formation penetration growth D.M. 1 65 1 30 2 2 Good Moderate Moderate D.M. 2 65 2 30 1 2 Good Moderate Moderate D.M. 3 65 2 30 2 1 Good Moderate Moderate D.M. 4 65 1 30 1 3 Good Moderate Moderate D.M. 5 65 1 30 3 1 Good Moderate Moderate D.M. 6 63 5 30 1 1 Good Moderate Moderate D.M. 7 60 5 30 2 3 Good Moderate Moderate D.M. 8 50 1 45 2 2 Good Good Good D.M. 9 45 5 45 2 3 Good Good Good D.M. 10 45 3 50 1 1 Good Good Good D.M. 11 40 3 55 1 1 Good Good Good D.M. 12 40 1 55 3 1 Good Good Good Table VIll(continue) (unit: w/w%) Delivery Medium Wheat bran Chitosan Wood Chitin Pharma Pellet Air Cellular sawdust media formation penetration growth D.M. 13 40 1 55 1 3 Good Good Good Control 1 90 2 5 2 1 Good Poor Poor Control 2 80 5 10 3 2 Good Poor Poor Control 3 70 3 25 1 1 Good Poor Poor Control 4 30 5 60 2 3 Poor Good nt Control 5 20 5 70 2 3 Poor Good nt Control 6 10 5 80 2 3 Poor Good nt nt: not tested Table IX (unit: Delivery Wheat Cliitosan Wood Chitin Pharia Initial cell no. of Cell NO. of WYE 20 Cellular growth Medium bran sawdust media WYE 20 or or WYE 324 after 3 WYE 324(cfu/g) months (cfu/g) at 4 C at 25 *C D. M. 1 65 1 30 2 2 1.2x] 07 1.0XI1 6 2.3 x 10' Good D.M. 2 65 2 30 1 2 l.9xI1 l.5x1 0 6 2.9x 10 5 Good D.M. 3 65 2 30 2 1 l.3,X10 7 1.4x 10' 2.5X105 Good D.M. 4 65 1 30132.1x]0 7 l.7x10 6 ".6x 105 Good D.M. 5 65 1 0 31 2.0x10 l.2x10l .x0 Good D.M. 6 6 3 5 3 0 11 2.7x 10' I. 1 X10 6 2.7x 10 5 Good D. M. 7 60 5 30 2 31.5x10 1.3x10 6 3.3xlI0W Good D.M. 8 50 1 45 2 2 1.6x10 8 2.1IX10 7 2. 1 X10 6 Good D. M. 9 45 5 45 2 -3 2.6x 10' 1.1x10 7 2.5x10I Good Table IX(continUe) (unit: Delivery Wheat Chitosan Wood Chitin Pharmna Initial cell no. of Cell NO. of WYE 20 Cellular growth Mediumn bran sawdust mnedia WYE 20 or or WYE 324 after 3 WYE 324(cfu/g) months (cfulg) at 4 C Iat 25 C D.M. 10 45 3 50 1 1 2. 1x 10' 1.0x10 7 .x 101 Good D.M. 11 40 3 55 1 1 1.9xI1 1.6x 10 7 5 x10 6 Good D.M. 12 40 1 55 31 1 1.8x 10 8 1.9x10 7 2.4x10I Good D.M. 13) 40 1 55 1 -3 2.2x 10' 1.3x0 19 1 Good WO 98/35017 PCT/KR98/00015 36 As shown in Tables VIII and IX, D.M. 1 to 13 were good and moderate in pellet formation, air penetration, and cellular growth, whereas Controls 1 to 6 with a different range of components were poor in pellet formation, air penetration, or cellular growth.

In addition, D.M. 1 to 13 pellets were coated with sporulation medium of YGM and determined the effects on cellular growth and viability. The results are shown in Table X.

Trable X (unit: Delivery Medium Wheat Chitosan Wood Chitin Pharma YGM* Initial cell no. of Cell No. of WYE 20 Cellular bran sawdust media WYE 20 or WYE or WYE 324 after 3 growth 324 (cfulg) months (cfulg) at 4 C at D.M. 14 65 1 30 2 2 0.2 1. 1 x10' 1.2x1 0 6 2.4x 10-' Good D.M. 15 65 2 30 1 2 0.5 1.4x 10' 1.4x1 0 6 2.9x 10' Good D.M. 16 65 2 30 2 1 1.0 1.6x10 7 1.6x10 6 2.6x 101 Good D.M. 17 65 1 30 1 3 1.5 2.Ox 10' .x0 4.2x 105 Good D.M. 18 65 1 30 31 1.8 2.2x 10' .x0 3.2x 10 5 Good D.M. 19 63 5 30 1 1 2.0 1.7x10 7 2.1IX10 6 3. 7x 10' Good D.M. 20 60 5 30 2 3 3.5 1.1X10 7 2.3x0 3.4x 105 Good D.M. 21 50 1 45 2 2 3.5 l.2xlO' 2.3x1 0 7 2.3x10 6 Good D.M. 22 45 5 45 2 3 2.5 1.6x 10' 2.1x0 2.5x10 Good D.M. 23 45 3 50 1 1 20 2.2x 10' I.3x10 7 3.2x 10 6 Good Table X (unit: Delivery MediUmn Wheat Chitosan Wood Chitin Pharma YGM* Initial cell no. of Cell No. of WYE 20 Cellular bran sawdust media WYE 20 or WYE or WYE 324 after 3 growth 324 (cfu/g) months (cfu/g) at 4IC at 25 IC D.M. 24 40 355 1 1 1.5 2.9x 10' 2.1Ix10 7 3.6x 10 6 Good D.M. 2S 40 1 55 -3 1 0.5 1.4x 10' 1.9x 10' 2.6x10 Good D.M. 26 40 1 55 1 3 0.2 1.2x10 8 1.3x10 7 2.9x 10 6 Good YGM*: wlw% of total weight.

_WO 98/35017 PCTII(R98/00015 39 As shown in Table IX and X, the cells of WYE 20 and WYE 324 were stabilized in D.M. 1 to 26 for a long period of time 3 months). The cells of WYE 20 and WYE 324 in D.M. 1 to 26 were shown 10' cfu/g which was required for the biocontrol activity after 3 months storage. In particular, when D.M. 14 to 26 coated with the medium of YGM were used, the stability of the cells of WYE 20 and WYE 324 was improved.

EXAMPLE VI Preparation of Biocontrol Agents in the Form of Suspension A delivery medium comprising 20g of pectin and 2g of colloidal chitin and the remaining water on the basis of the total volume of 1.0 liter distilled water was autoclaved at 121 °C for 15 minutes. Preparation of a biocontrol agent in the form of suspension was achieved by incorporating Streptomyces sp. WYE 20 or WYE 324 as described in "Materials and Methods", into the resulting autoclaved delivery medium (1.2 x 10' cfu/ml).

The resulting product was kept at 4 The form of biocontrol agent was suitable for the treatment of plant seeds and seedling beds by in-furrow application, or spraying after dilution with water.

EXAMPLE VII Antifungal Activity Assay In vivo biocontrol assays were carried out to determine the efficacy of biocontrol agents prepared in Example V and Example VI. The biocontrol agents were tested for their ability to reduce fungal diseases and enhance plant growth by treating plants, plant seeds, plant roots, seedling beds, pots, potting mixtures, or soil.

Biocontrol Activity to Rhizoctonia Damping-off of Cucumber (Cucumis sativus A biocontrol assay was carried out to test the efficacy of WYE 20 as a biocontrol agent in inhibiting Rhizoctonia damping-off of cucumber.

WO 98/35017 PCTIKR8/00015 Cucumber seeds were treated with 20 ml of WYE 20 (1.2 x 107 cfu/ml) prepared in Example VI by immersing seeds therein for three hours and then being seeded in pots. Seeds were immersed for three hours in sterilized distilled water and a delivery medium, respectively; each resulting seed was used as a control.

Hortus (England) was used as a potting mixture. The potting mixture was autoclaved at 121 "C for 60 minutes, and placed for 12 hours at 25 and then autoclaved at 121 °C for 60 minutes again. This autoclaving cycle was repeated three times.

Rhizoctonia solani was cultured on PDB (Potato dextrose broth: Difco) at 25 °C for 14 days, harvested and mixed with autoclaved potting mixture so as to obtain an approximately 60% disease incidence. The cucumber seeds were treated with biocontrol agent and each control seed was seeded in pots containing the potting mixture into which Rhizoctonia solani had been artificially inoculated.

Disposable paper cups (diameter of 9cm) were used as seedling pots. Six cups containing three seeds per cup were prepared in each test. The cups were placed in a random block design in a glasshouse. Moisture was kept between and 60% and additional water was supplied as needed. The temperature was maintained between 25 °C and 30 "C in daylight.

The results of emergence and occurrence of damping-off were periodically recorded. The final results are shown in Table XI.

WO 98/35017 PCT/I8/00015 41 Table XI Emerged Healthy Fresh Weight No. Of Cucumber Seeds Plants (g)/above Treatments Cucumber ground Seeds 7 days 14 days 18 days No pathogen 18 18b" (100) 18b' (100) 2.61b" control Rhizoctonia solani 18 7a (39) 7a (39) 1.73a control Rhizoctonia solani Liquid D.M. 18 8a (44) 8a (44) 1.75a treatment Rhizoctonia solani Liquid D.M. of 18 18b (100) 18b(100) 2.51b WYE xMeans in a column followed by the same letter are not significantly different at the P 0.05 level. Delivery Medium.

As shown in Table XI, cucumber seeds treated with WYE 20 in delivery medium as a biocontrol agent did not show Rhizoctonia damping-off of cucumber.

In addition, cucumber plants from control seeds showed severe growth retardation resulting from Rhizoctonia solani, whereas plants from treated seeds did not. The results showed that WYE 20 was effective in controlling Rhizoctonia solani and in enhancing plant growth.

Biocontrol Activity Against Powdery Mildew of Cucumber (Cucumis sativus A biocontrol assay was carried out to test the efficacy of WYE 324 (1.2 x 10 7 cfu/ml) in the biocontrol agent prepared in Example VI in inhibiting powdery wO 98/35017 PCTIKR98/00015 42 mildew of cucumber plants in pots.

Disposable paper cups (diameter of 9cm) were used as seedling pots. One cucumber seed was seeded in each cup containing potting mixture consisting of agricultural field soil and Hortus (England) (4:1 v/v ratio). The cups were placed in a glasshouse. Additional water was supplied as needed and the temperature was maintained between 25 C and 30 *C in daylight.

Twenty ml of the biocontrol agent containing WYE 324 were sprayed on one cucumber plant (14-day-old) at the beginning of the assay and sprayed one more time after a week. Plants sprayed with the same amounts of water were used as a control. Three cucumber plants (one cucumber plant per cup) of each treated group and the control group were prepared in the assay.

To naturally induce powdery mildew of cucumber caused by Sphaer-ohecafuliginea, three cups per group and three diseased cucumber plants (one plant per cup) were used and set in a random block arrangement in a glasshouse. Moisture was kept between 70% and 90% and additional water was supplied as needed. The temperature was maintained between 25 °C and 30 "C in a glasshouse. The incidence of cucumber powdery mildew was investigated. The experiment was continued for 2 weeks and the assay was repeated. The results are shown in Table XII.

Table XII Incidence of Powdery Mildew of Cucumber Treatments Experiment 1 Experiment 2 Control WYE 324 Treated As shown in Table XII, powdery mildew was not detected in cucumber plants sprayed with the biocontrol agent containing WYE 324; whereas the disease was observed in control plants. The results showed that WYE 324 was WO 98/35017 PCT/KR98/00015 43 effective in controlling powdery mildew of cucumber (Cucumis sativus Biocontrol Activity of Streptomyces sp. WYE 20 or WYE 324 to Rhizoctonia Brown Patches of Golf Green Creeping Bentgrass A biocontrol assay was carried out to test the efficacy of Streptomyces sp.

WYE 20 and WYE 324 in inhibiting Rhizoctonia brown patch of golf green creeping bentgrass.

Streptomyces sp. WYE 20 and WYE 324 produced by liquid culture as described in "Materials and Methods" above were used in this assay. The cell number of Streptomyces sp. WYE 20 or WYE 324 was adjusted to 2.0 x 105 cfu/ml. The assay was carried out on each block treated with WYE 20 or WYE 324, or control. Four blocks (1.5m x 1.5m) of each treated group and control group were prepared on a golf green. The assay was carried out in a random block design. Strain WYE 20 or WYE 324 was sprayed every 7 10 days in the experimental blocks from June 25, 1996 to August 23, 1996. Two and a half liters of WYE 20 or WYE 324 per block were used each time; whereas the control blocks were provided with the same amount of water. No chemical fungicides were used in the experimental period. The incidence of Rhizoctonia brown patches was periodically recorded and shown in Table XIII (Site 1) and Table XIV (Site 2).

As shown in Table XIII (Site 1) and Table XIV (Site the incidence of Rhizoctonia brown patches was dramatically reduced in blocks treated with WYE or WYE 324. The results showed that WYE 20 and WYE 324 were effective in inhibiting Rhizoctonia brown patches of golf green creeping bentgrass.

WO 98/35017 PCT/KR98/00015 44 Table XIII (Site 1) Incidence of Rhizoctonia Brown Patches Treatments 7/25/96 8/6/96 8/23/96 Control 10.18a 14.6ax 59a' WYE 20 treated 3.38b 9.2b 24b WYE 324 treated O.OOc 5.2c 24b 'Means in a column followed by the same letter are not significantly different at the P 0.05 level.

Table XIV (Site 2) Incidence of Rhizoctonia Brown Patches Treatments 7/25/96 8/6/96 8/19/96 Control 5.6a" 9.3a 28.3a' WYE 20 treated O.Ob 0.8b 5.3b WYE 324 treated O.Ob 0.Ob 0.7c xMeans in a column followed by the same letter are not significantly different at the P 0.05 level.

Biocontrol Activity to Rhizoctonia Large Patches of a Fairway Turfgrass (Zoysia japonica).

WO 98/35017 PCT/IMR8/00015 A biocontrol assay was carried out to test the efficacy of biocontrol agents including Streptomyces sp. WYE 20 or strain IBT 678 prepared in Example VI in inhibiting Rhizoctonia large patches of fairway turfgrass (Zoysia japonica). The cell number of Streptomyces sp. WYE 20 or strain IBT 678 in a biocontrol agent was adjusted to 2.0 x 106 cfu/ml.

Soils infested with Rhizoctonia solani AG 2-2 were inoculated into a fairway turfgrass (Zoysia japonica) and then planted in pots (diameter of Two hundred ml of the biocontrol agent were treated once per week for two weeks and three weeks in 1996 and 1997, respectively. Pots treated with the same amounts of water were used as a control group. Three pots per group were used and set in a random block arrangement. To naturally induce disease, the pots were placed near the fairway of a golf course from the beginning of September to the beginning of October, 1996 as well as from the beginning of March to the end of June, 1997, respectively. The incidence of Rhizoctonia large patches was investigated. Large patches were not detected in pots treated with the biocontrol agent containing WYE 20; whereas the disease was observed in control and IBT 678 treated pots as shown in Table XV.

Table XV Incidence of Large patches of Zoysia Treatments japonica Fall 1996 Spring 1997 Pathogen Control Pathogen WYE 20 Treated Pathogen IBT 678 Treated Not tested As shown in Table XV. the results showed that WYE 20 was effective in controlling Rhizoctonia large patches of a fairway turfgrass (Zoysiajaponica).

WO 98/35017 PCT/KR98/00015 46 Biocontrol Activity Against Phytophthora Blight of Pepper Plant(Capsicum annuum L.) A biocontrol assay was carried out to test the efficacy of WYE 20 or WYE 324 in the biocontrol agent of Example VI in inhibiting Phytophthora blight of pepper seedlings in pots.

Pepper seeds were submerged in sterilized water for 2 days and treated with WYE 20 or WYE 324 in the biocontrol agent of Example VI by submerging them for 18 hours (treating group). The cell numbers of Streptomyces sp. WYE and WYE 324 were adjusted to 1.2 x 107 cfu/ml and 1.7 x 107 cfu/ml, respectively. Pepper seeds were submerged in sterilized water for 2 days and then for 18 hours. They were used as a control group. On the other hand. pepper seeds submerged in sterilized water for 2 days were replaced in the delivery medium of Example VI for 18 hours. They were used as the other control group.

As described in the assay with cucumber above, Phytophthora capsici cultured in PDB (Potato dextrose broth: Difco) at 25 °C for 14-21 days was harvested and inoculated into a sterilized potting mixture (Hortus, England) to obtain an infested potting mixture with a disease incidence of 80% of Phytophthora blight for control pepper seedlings. The resulting potting mixture infested with Phytophthora capsici was used to determine the efficacy of WYE 20 or WYE 324 in the delivery medium in inhibiting phytophthora blight of pepper seedlings in the assay.

Disposable paper cups (diameter of 9.0cm) were used as seedling pots.

Fourteen cups containing three seeds per cup were prepared in each test.

Experiments were performed in a glasshouse at a temperature of 25 °C to 32 °C in daylight. In the glasshouse, pots were set in a random block arrangement. The moisture was maintained at 80% and additional water was sprayed on the top of the pots as needed.

The emergence and incidence of Phytophthora blight of pepper plants were recorded as shown in Table XVI.

Table XVI No. of Emerged Pepper seedlings showing Phytophthora No. of Planted Pepper Seeds blight (disease incidence: Treatents Pepper Seeds (Emergence: 14 days 14 days 18 days No pathogen 42 37(88a') 0( 00x O( Ocx) Phytophthora capsici 42 34(8 1Ia) 28(82a) -33(9 7a) Control Phytophthora capsici 42 35 (8 3a) 29(83a) 34(97a) liquid D.M. Treated Phytophthora capsici 42 34(8 1Ia) 12(35b) 26(76b) WYE 20 Treated Phytophthora capsici 42 36(86a) 22(61 c) 28(78b) WYE 324 Treated 'Means in a column followed by the same letter are not significantly different at the P =0.05 level.

WO 98/35017 PCT/KRM8/00015 48 As shown in Table XVI, there was a significant reduction in Phytophthora blight of pepper plants in the seeds treated with WYE 20 or WYE 324 as compared to the control group. The result showed that WYE 20 and WYE 324 of the present invention were effective in controlling Phytophthora blight of pepper plants.

An Assay to Determine Plant Growth Enhancement Using Pepper Seedlings A seedling assay was carried out to determine the efficacy of WYE and WYE 324 in a biocontrol agent of the present invention in enhancing the growth of pepper plants.

Pepper seeds submerged in sterilized water for 2 days were treated with WYE 20 or WYE 324 in a biocontrol agent as prepared in Example V 22) (1000 seeds per 4g biocontrol agent). The cell numbers of Streptomyces sp.

WYE 20 and WYE 324 prior to seed treatment were adjusted to 1.2 x 10 cfu/ml and 1.7 x 107 cfu/ml, respectively. Pepper seeds were submerged in sterilized water and used as a control group (Control Meanwhile, pepper seeds were treated with the delivery medium (1000 seeds per 4g D.M.22) not containing WYE 20 or WYE 324 and used as the other control group (Control 2).

The planting of the pepper seeds was carried out in the same way as the cucumber seedlings shown above.

Disposable paper cups (diameter of 9.0cm) were used as seedling pots.

One hundred and twenty one cups containing one seed per cup were prepared in each test. Experiments were performed in a glasshouse at a temperature of 25 °C to 32 "C in daylight. In the glasshouse, pots were set in a random block arrangement. The moisture was maintained in a range of 40% to 60% and additional water was sprayed on the top of the pots as needed. Fifteen ml of cell culture broth of WYE 20 or WYE 324 obtained in "Materials and Methods" were inoculated into the seedling pot treated with the biocontrol agent after 4 weeks of cultivation. The same amount of water was applied to the control groups. The experiments were continued for 9 weeks and the results were shown in Table WO 98/35017 PCT/KR98/00015 49

XVII.

Table XVII Treatments Pepper Seedlings Plant Height (cm) (average) Control 1 121 23.7a Control 2 121 24.5a WYE 20 Treated 121 27.4b WYE 324 Treated 121 28.6b,c xMeans in a column followed by the same letter are not significantly different at the P 0.05 level.

As shown in Table XVII, there was a significant enhancement in the growth of the plants from the pepper seeds treated with WYE 20 or WYE 324 as compared to those plants germinated from control seeds. This indicates that WYE or WYE 324 of the present invention is highly effective in enhancing the growth of pepper plants.

A Biocontrol Assay Using Pepper Seedlings in Agricultural Fields A biocontrol assay was carried out to determine the efficacy of a biocontrol agent of the present invention for controlling Phytophthora blight and for enhancing plant growth in agricultural fields.

Pepper seeds submerged in sterilized water for 2 days were immersed in a biocontrol agent prepared in Example VI for 3 hours. The seeds were treated with a biocontrol agent (D.M.22) (1,000 seeds per 4g) prepared in Example V.

The cell numbers of Streptomyces sp. WYE 20 and WYE 324 prior to seed treatment were adjusted to 1.2 x 107 cfu/ml and 1.7 x 10' cfu/ml, respectively.

Pepper seeds were submerged in sterilized water for 2 days and 3 hours -WO 98/35017 PCeTKR98/00015 again. Thus the pepper seeds were treated with the same delivery medium not containing WYE 20 or WYE 324 and used as a control group. Each seed was planted in seedling beds. Water was sprayed as needed and a temperature of between 20 "C and 35 *C was maintained. When seedlings were grown to 1.5 to 2.0cm in height, the pepper seedlings were transplanted into a seedling tray consisting of 25 seedling holes (5cm x 5cm, 6cm depth) containing a mixture of fine sandy potting soil and WYE 20 or WYE 324 in the delivery medium (0.1 g per seedling hole). Control pepper seedlings were transplanted into seedling holes containing the same amount of sandy potting soil and the same amount of delivery medium only. These seedling pots were incubated in a greenhouse at a temperature of 18 "C to 35 "C and additional water was supplied as needed. In the greenhouse, pots were set in a random block arrangement. After 11 weeks of growth in the greenhouse, each plant was transplanted to agricultural fields.

Before a week of transplantation, 10 ml culture broth of WYE 20 or WYE 324 (1.2-1.7 x 105cfu/ml) were added per seedling hole of treating group. The same amount of water was supplied to control seedling pots.

The disease incidence and the growth of the transplanted peppers were observed periodically and the mean was recorded as shown in Table XVIII (Field 1) and XIX (Field 2).

Table XVIII (Field 1) Incidence of phytophthora Blight Plant Height (cm) No. of Transplanted PatHih( Treatments at 62 days after Pepper Plants 62 days after 101 days after transplantation transplantation transplantation Control 1,340 78.5a 28.4ax 98.6a x WYE 20 Treated 1,340 86.0b 23.5b 89.2b WYE 324 Treated 1,340 90.5c 8.7c 76.1c 'Means in a column followed by the same letter are not significantly different at the P 0.05 level.

-WO 98/35017 PCTAKR98/oom1 52 Table XIX (Field 2) Incidence of Phytophthora Blight No. of Treatments Transplanted 82 days after 101 days after Pepper Plants Peprs____ transplantation transplantation Control 860 26a" 42.4a' WYE 20 Treated 860 Ob 7.6b WYE 324 Treated 860 Ob xMeans in a column followed by the same letter are not significantly different at the P 0.05 level.

As shown in Table XVIII and XIX, there was a significant enhancement in the growth and reduction of Phytophthora blight in the plants from the pepper seeds treated with WYE 20 or WYE 324 as compared to those plants germinated from control seeds. This indicates that WYE 20 and WYE 324 of the present invention are highly effective in controlling Phytophthora blight and in enhancing plant growth of pepper in agricultural fields.

Having provided examples of embodiments of the present invention and preferred embodiments, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the present invention and its broader aspects. Therefore, numerous variations, modifications, and embodiments are to be regarded as being within the true spirit and scope of the present invention.

-WO 98/35017 PCT/KR98/00015 53

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Claims (24)

1. A biologically pure culture of a microorganism of Streptomyces sp. WYE 20 and Streptomyces sp. WYE 324, having the identifying characteristics of KCTC 0341BP and KCTC 0342BP, respectively.

2. Antifungal biocontrol agent including a microorganism of a biologically pure culture of Streptomyces sp. WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP).

3. The microogransim of Claim 1 and 2 wherein the microorganism is able to protect susceptible plants against fungal infection.

4. The microorganism of Claim 3 wherein protection is conferred against fungal pathogens that cause seedling diseases including damping-off and root rot as well as foliar diseases including powdery mildew of cucumber, Phytophthora blight of pepper, or Rhizoctonia Sbrown/large patches of turfgrasses.

5. Antifungal biocontrol agent of Claim 2 wherein the biocontrol agent comprises a biologically pure culture of Streptomyces sp. WYE 20 or WYE 324; and a delivery medium. 15

6. Antifungal biocontrol agent of Claim 5 wherein the delivery medium consists of 40 to 65 w/w% of wheat bran, 1 to 5 w/w% of chitosan, 30 to 55 w/w% of wood sawdust, 1 to 3 w/w% of chitin, and 1 to 3 w/w% of Pharmamedia on the basis of the total weight of the delivery medium.

7. Antifungal biocontrol agent of Claim 6 wherein the delivery medium further consists of 0.2 to 3.5 w/w% of sporulation medium.

8. Antifungal biocontrol agent of Claim 6 and 7 wherein the biologically pure culture of Streptomyces sp. WYE 20 or WYE 324 comprises 105-10' colony forming units per gram of delivery medium.

9. Antifungal biocontrol agent of Claim 5 wherein the delivery medium consists of 1.0 to 3.0 w/w% of pectin and 0.1 to 0.6 w/w% of colloidal chitin in water. Antifungal biocontrol agent of Claim 9 wherein the biologically pure culture of Streptomyces sp. WYE 20 or WYE 324 comprises 105-101o colony forming units per gram of delivery medium.

CE00372179.3 61

11. A method of manufacturing an antifungal biocontrol agent comprising an incubation process for the production of a biologically pure culture of Streptomyces sp. WYE20 (KCTC 0341BP) or WYE324 (KCTC 0342BP) and a downstream process comprising lyophilization of the harvested pure culture of Streptomyces sp. WYE 20 OR WYE 324.

12. A method of manufacturing an antifungal biocontrol agent comprising an incubation process for the production of a biologically pure culture of Streptomyces sp. WYE20 (KCTC 0341BP) or WYE324 (KCTC 0342BP) and a downstream process comprising incorporation of the harvested pure culture into a proper delivery medium.

13. A method of manufacturing an antifungal biocontrol agent according to Claims 11 or 12 10 wherein said incubation process comprises incubation at 130 to 300 rpm at 25 0 C to 33 0 C for 3 to 7 days.

14. A method of manufacturing an antifungal biocontrol agent wherein the method comprises the steps of: o preparation of delivery medium consisting of 40 to 65 w/w% of wheat bran, 1 to 15 w/w% of chitosan, 30 to 55 w/w% of wood sawdust, 1 to 3 w/w% of chitin, and 1 to 3 Sw/w% of Pharmamedia on the basis of the total weight of the delivery medium; o: autoclaving the resulting delivery medium; incorporation of Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) into the delivery medium; incubating incorporated cells of Streptomyces sp WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) at 25 0 C to 33°C for 5 to 14 days; and aseptically drying the resulting product.

A method of manufacturing an antifungal biocontrol agent according to Claim 14 wherein the method comprises aseptically blending the resulting dried product.

16. A method of manufacturing an antifungal biocontrol agent according to Claim 14 R, herein said preparation of delivery medium comprises palletizing the delivery medium and Sc ing the resulting pellet with 0.2 to 3.5 w/w% of sporulation medium. CE00372179.3 62

17. A method of manufacturing an antifungal biocontrol agent according to Claim 14 wherein Streptomyces sp. WYE 20 or WYE 324 for incorporation comprises 10 5 -10' 0 colony forming units per gram of delivery medium.

18. A method of manufacturing an antifungal biocontrol agent according to Claim 14 wherein said autoclaving is carried out at 121 0 C for 30 to 40 minutes.

19. A method of manufacturing an antifungal biocontrol agent wherein the method comprises the steps of: preparation of delivery medium consisting of 1.0 to 3.0 w/w% of pectin, 0.1 to 0.6 w/w% of colloidal chitin, and the remaining water; and autoclaving the resulting delivery medium; and incorporation of Streptomyces sp. WYE

20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) into the delivery medium. A method of manufacturing an antifungal biocontrol agent according to Claim 19 wherein Streptomyces sp. WYE 20 or WYE 324 for incorporation comprises 10"- 10 l o colony forming units per gram of delivery medium.

21. A method of use of an antifungal biocontrol agent of Claims 2 to 10 wherein the use comprises coating, mixing, spraying or in-furrow application to plant seeds, potting mixtures, growing plants, or soils.

22. A biologically pure culture of Streptomyces substantially as herein described with reference to the accompanying examples. 0

23. An antifungal biocontrol agent substantially as herein described with reference to the S accompanying examples. C 0 o°•0. CE00372179.3 62a

24. A method of manufacturing an antifungal biocontrol agent substantially as herein described with reference to the accompanying examples. A method of use of an antifungal biocontrol agent substantially as herein described with reference to the accompanying examples. Dated this 8th day of February 2001 Hyung Won Suh by his attorneys Freehills Carter Smith Beadle a a a. a a a a. a a a a. a a. a a a a a